DESCRIPTION Two intriguing recent observations in stroke research are that, 1) the outer region of an ischemic infarct, the ischemic penumbra, is potentially salvageable tissue; and 2) waves of infarct-associated spreading depression invade the penumbra, and their frequency and severity correlate with the ultimate extent of structural damage. A key step in understanding why an ischemic infarct expands might be to establish why and how waves of spreading depression are generated within ischemic tissue. The PI has demonstrated that an intact gap junctional network is required for the propagation of spreading depression supporting the notion that spreading depression is an in vivo correlate to gap junction-mediated astrocytic calcium signaling. Imaging of calcium signaling among a population of ischemic astrocytes might therefore provide a powerful tool for analysis of interactions between dying and viable cells. On this basis, the PI postulates that aberrant calcium signaling initiated in dying astrocytes can cause the death of otherwise viable neighbors. Over the last few years the PI's lab has developed a number of methodologies which have allowed: 1) the characterization of gap junctional signaling on a cell-to-cell level 2) the manipulation of the degree of gap junctional coupling by connexin over-expression, 3) influence the resistance of cultured cells to ischemia, by bcl-2 overexpression, 4) the block of the propagation of SD by gap junction blockers, and 5) the evaluation of gap junctional coupling in the live rat. In preliminary studies, it has been established that astrocytic gap junctions remain functional during the process of cell death, that spontaneous calcium activity is initiated during ischemia, and that the extent of injury is a function of the degree of gap junctional coupling. It is now proposed to exploit these advances by applying them to a previously intractable problem, that of the role of signaling across gap junctions in acute brain injury. Experimental questions will include: 1. Are astrocytic gap junctions functional during the process of cell death? 2. Can dying astrocytes cause death of otherwise viable neighboring cells? 3. Is focal ischemia associated with a decrease in gap junctional function intake live rat brain? 4. Will manipulations of junctional conductance affect the outcome of ischemia?